MICROGONOTROPENS AND THEIR INTERACTIONS WITH DNA .1. SYNTHESIS OF THETRIPYRROLE PEPTIDES DIEN-MICROGONOTROPEN-A, DIEN-MICROGONOTROPEN-B, AND DIEN-MICROGONOTROPEN-C AND CHARACTERIZATION OF THEIR INTERACTIONS WITH DSDNA

Exploration of the novel idea to employ a pyrrole nitrogen of a tripyr role peptide minor groove binding agent to carry catalytic entities to the phosphates and major groove of DNA has been initiated with the sy nthesis of dien-microgonotropen-a, -b, and -c (5a, 5b, and 5c). Replac ing the carboxyl terminal amidine and amino terminal formyl functional ities of distamycin (Dm) by CH2N(CH3)2 and acetyl substituents, respec tively, provides 2, which has greater stability in water than does Dm. The synthetic design allows the N-methyl substituent on the central p yrrole of 2 to be replaced by connectors terminating in a dien ligand [-(CH2)3N{(CH2)3N(CH3)2}2 (5a), -(CH2)4N{(CH2)3N-(CH3)2}2 (5b), -(CH2) 5N{(CH2)3N(CH3)2}2 (5c)]. The binding of 2 is about 20-fold weaker tha n the binding of 5a, 5b, and 5c to calf thymus DNA, poly(dA-dT), and p oly(dI-dC) due to the contribution of the polyamine substituents of th e latter. The specificity and affinity of binding of 5a, 5b, and 5c to the 5'-[P-32] 167-bp EcoRI/RsaI restriction fragment of pBR322 was de termined by DNase I footprint analysis. Specific inhibition of cleavag e was observed at each of the four potential A+T-rich binding sites af ter preincubation with 5a, 5b, and 5c at concentrations as high as 50 muM. At 250 muM, binding at short heteropolymeric A+T secondary sites distal to the cluster of A+T-rich primary binding sites was observed. At such higher concentrations of 5a, 5b, and 5c, increased rates of en zymatic cleavage at specific sequences were observed. DNase I footprin ting analysis of the 3'-labeled fragment provided complementary result s. Electrophoretic migration of HaeIII restriction digest fragments of phiX-174-RF DNA after preincubation with 5a, 5b, and 5c was used to a ssess induction of gross conformational changes in DNA molecules. As t he concentration of the agents increases, the effect of the agents in changing the conformation of larger DNA fragments decreases in the ord er 5c > 5b > 5a >> Dm > Hoechst 33258. The electrophoretic mobilities of smaller DNA fragments are unaltered in the presence of the various agents. The dien-microgonotropens are much more effective in inducing changes than the sum of the Dm and bis[3-(dimethylamino)propyl]methyla mine parts. This is due to the unique relationship between the minor g roove binding portion of the dien-microgonotropens and the accompanyin g electrostatic complexing of the covalently attached dien moiety to t he phosphodiester backbone of DNA.